We observed that the homeotic gene, HOXA5 has decreased expression in breast cancer cell lines and in primary breast tumors, when compared to normal mammary epithelial cells (HMEC). We have previously demonstrated its ability to modulate the expression of p53 and the progesterone receptor. Recently, using two separate technologies, we have identified genes that are responsive to, or interact with HOXA5. 1) By performing microarray analysis of Saccharomyces cerevisiae that express human HOXA5, we have identified novel putative target genes that may be regulated by HOXA5 protein. One of the genes that is up-regulated by HOXA5 in yeast is a DNA mismatch repair gene whose human paralog was identified as MutL-1 (hMLH1). The promoter region of hMLH1 contains four putative HOXA5 binding sites. We have now demonstrated that over-expression of HOXA5 up-regulates hMLH 1 in a breast cancer cell line, MCF- 7. We will now evaluate the regulation of hMLH1 by HOXA5 and its role in the critical function of maintaining the integrity of the cellular genome. 2) Using yeast two-hybrid analysis, we have identified an anti-apoptotic gene, Twist, as an interactor of HOXA5 protein. We have shown that: a) expression of Twist in a breast cancer cell line caused a decrease in p27[Kip1] levels while increasing the cell's ability to survive during serum starvation and, b) in functional assays, HOXA5 can partially restore the suppressive effects of Twist on p53 target genes. We will fully characterize the nature of the HOXA5/Twist interaction. In addition to studying the biochemical interactions between these proteins, we would like to shed light on the biology of HOXA5 in the breast. Loss of expression of HOXA5 occurs in 60% of breast cancers. However, little is known regarding its expression throughout normal breast development, or its relationship with other prognostic factors. 3) We will use immunohistochemistry (IHC) to characterize HOXA5 expression in normal breast during development from pre-pubertal to postmenopausal stages and in cancer. By examining breast tumor arrays by IHC, we would like to determine if loss of HOXA5 correlates with high grade breast tumor formation. Thus, we propose to examine, in depth, the role of HOXA5 in breast cancer. These studies will yield new insights into the tumor suppressor role of HOXA5 and further elucidate the molecular functions of HOXA5 in breast tumorigenesis.